The present invention relates to novel non-targeting dithiocarbamate-containing compositions. In one aspect, the present invention relates to non-targeting dithiocarbamate-containing compositions wherein the dithiocarbamate is non-covalently associated with a macromolecule other than an antibody. Preferably, the macromolecule is non-immunogenic. In another aspect, the present invention relates to non-targeting dithiocarbamate-containing compositions wherein the dithiocarbamate is covalently crosslinked with a macromolecule other than an antibody that is preferably non-immunogenic. In yet another aspect, the present invention relates to diagnostic and therapeutic methods employing the novel non-targeting dithiocarbamate-containing compositions described herein.
In 1984, Jolly et al., demonstrated the protection of reperfused myocardial tissue with the combinational use of superoxide dismutase and catalase (see, for example, Jolly et al., Cir. Res., 57:277, 1984). This observation implied that oxygen-derived free radicals are a cause of the reperfusion injury to the hypoxic myocardium. It is now known, however, that the phenomenon of ischemia/reperfusion injury is not restricted to the myocardium. Instead, ischemia/reperfusion injury is viewed as a general damaging event in any tissue or organ (such as brain, liver or kidney) subjected to a critical period of ischemia followed by perfusion with oxygenated whole blood.
Ischemia/reperfusion injury therefore results from the reintroduction of molecular oxygen at the time of organ reperfusion or restoration of the circulation. While the delivery of dissolved molecular oxygen sustains cellular viability, it also provides oxygen as a substrate for numerous enzymatic oxidation reactions that produce reactive oxygen species which cause oxidative damage, a phenomenon referred to as the xe2x80x9coxygen paradoxxe2x80x9d (see, for example, Hearse et al., in J. Mol. Cell. Cardiol., 10:641, 1978). Oxygen, a gaseous molecule essential for normal cellular metabolism, can, under certain conditions, be deleterious to life. The cell defends itself against oxidative insults through its antioxidant mechanisms including superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase and cellular antioxidants including glutathione, ascorbate and a-tocopherol (see, for example, Chan, in Stroke, 27:1124-29, 1996). However, when reactive oxygen species are generated at a rate that exceeds the capacity of the cell to defend itself against the resulting oxidative stress (such as in ischemia/reperfusion insults), the cell is irreversibly damaged, resulting in necrotic cell death or ischemic cell death.
Although the exact mechanism by which oxygen induces ischemic cell death is not yet clear, it is well known that reactive oxygen species cause a wide range of tissue damage. The hydroxyl radical (.OH), the most potent oxidant, is capable of initiating lipid peroxidation, causing protein oxidation and DNA damage in cells (see, for example, Lai and Piette, in Biochem. Biophys. Res. Commun., 78:51-59, 1977 and Dizdaroglu and Bergtold, in Anal. Biochem., 156:182, 1986). Albeit less reactive, superoxide anion radicals (.O2), on the other hand, participate in a repertoire of oxidative reactions which generate hydrogen peroxide and hydroxyl radical as follows:
.O2xe2x88x92+.O2xe2x88x92xe2x86x92H2O2xe2x80x83xe2x80x83(1) 
.O2xe2x88x92+H2O2xe2x86x92.OH+OHxe2x88x92+O2xe2x80x83xe2x80x83(1) 
Reaction (1) is catalyzed by SOD, while reaction (2) proceeds rapidly in the presence of trace iron metal (see, for example, Haber and Weiss, in Proc. R. Soc. Ser. A., 147:332, 1934). Superoxide anion radical is known to liberate iron from ferritin (see, for example, Wityk and Stem, in Crit. Care Med., 22:1278-93, 1994) which further facilitates the iron-catalyzed Fenton reaction in the reoxygenated tissue, generating damaging hydroxyl radicals, as shown in reactions (3) and (4), see, for example, Halliwell and Gutteridge, in Halliwell and Gutteridge. Free Radicals in Biology and Medicine, 2nd edition. Oxford: Clarendon Press, 15-19 (1989):
Fe3++O2xe2x88x92xe2x86x92Fe2++O2xe2x80x83xe2x80x83(3) 
Fe2++H2O2xe2x86x92.OH+OHxe2x88x92+Fe3+xe2x80x83xe2x80x83(4) 
In addition to reactive oxygen species, reactive nitrogen species such as nitric oxide (.NO) have also been observed to be excessively produced in ischemia/reperfusion organs (see, for example, Faraci and Brian, in Stroke, 25: 692-703, 1994). .NO is synthesized from the terminal guanidino nitrogen atom of L-arginine by nitric oxide synthase (NOS). Three different isoforms of NOS have been isolated, cloned, sequenced and expressed (see, for example, Nathan, in FASEB J., 6:3051-3064, 1992), i.e., eNOS, NNOS and iNOS. The eNOS (endothelial cell derived) and nNOS (neuronal cell derived) are expressed constitutively, and both enzymes require an increase in intracellular calcium for activation.
Under physiological conditions, a low output of .NO is released continuously from eNOS in endothelial cells and from nNOS in neuronal cells. This .NO serves to dilate blood vessels and, in concert with vasoconstrictor catecholamines, regulate blood flow and blood pressure. On the other hand, a high output of .NO is produced by the inducible, calcium-independent NOS (INOS) isoform upon activation with cytokines or endotoxin (see, for example, Moncada and Higgs, in New Engl. J. Med., 329:2002-2012, 1993). iNOS is expressed in numerous cell types, including endothelial cells, smooth muscle cells, microglial cells and macrophages. Abnormally elevated levels of nitric oxide have recently been associated with ischemia/reperfusion injury (see, for example, Kumura et al., in J. Cereb. Blood Flow and Metab., 14:487-491, 1994; Iadecola et al., J. Cereb. Blood Flow and Metab., 15:378-384, 1995).
In the central nervous system, nitric oxide has been discovered to function as both a neurotransmitter and a neurotoxin (see, for example, Faraci and Brian, in supra.). It mediates N-methyl-D-aspartate (NMDA) excitotoxicity. Elevated .NO levels in the brain have been measured during ischemia using an .NO electrode (for example, see Malinski et al., J Cereb.Blood Flow Metab., 13:355-358,1993), and by electron paramagnetic resonance spin trapping (for example, Sato et al., Biochim. Biophys. Acta, 1181:195-197, 1993). .NO levels began to increase within minutes after the onset of ischemia, presumably reflecting an increased activity of constitutive .NO synthase. However, as ischemia continues, .NO levels fall slowly but then increase again during reperfusion (see, for example, the recent review by Dawson and Dawson in Cerebrovascular Disease, H. Hunt Batjer, ed., Lippincott-Raven Publishers, Philadelphia, pp. 319-325 (1997)). The expression of iNOS gene was demonstrated in the rat brain to begin at 12 hours and peaked at 48 hours following the cerebral ischemia (Iadecola et al., supra).
.NO may have both beneficial and detrimental effects during cerebral ischemia. Increased .NO production during ischemia may be protective because .NO increases cerebral blood flow and inhibition of aggregation and adherence of platelets or leukocytes (see, for example, Samdani et al., in Stroke 28:1283-1288 (1997)). On the other hand, excessive .NO production during reperfusion is cytotoxic, either directly or after recombination with superoxide anion radical to form peroxynitrite according to reactions (5)-(7), as follows:
.O2xe2x88x92+.NOxe2x86x92ONOOxe2x88x92xe2x80x83xe2x80x83(5) 
ONOOxe2x88x92+H+xe2x86x92ONOOHxe2x80x83xe2x80x83(6) 
ONOOHxe2x86x92[.OH]+.NO2xe2x80x83xe2x80x83(7) 
It has been demonstrated in cell-free systems that superoxide anion radical chemically reacts with nitric oxide to form the toxic anion, peroxynitrite, ONOOxe2x88x92 (reaction (5), see, for example, Beckman et al., in Proc.Natl. Acad. Sci., USA 87:1620-1624, 1990). The rate constant for the reaction of nitric oxide with superoxide anion is 6.7xc3x97109 Mxe2x88x921Sxe2x88x921 (see, for example, Huie and Padmaja, in Free Radical Res. Commun., 18:195-199, 1993) which is three times faster than that for the dismutation of superoxide anion radicals by superoxide dismutase (reaction (1); 2-3xc3x97109 Mxe2x88x921Sxe2x88x921) (see, for example, Hassan et al., in Free Radical Biol. Med., 5:377-385, 1988). At physiological pH, peroxynitrite is essentially protonated (reaction (6)), which decomposes readily to form a hydroxyl radical-like species (i.e., xe2x80x9c[.OH]xe2x80x9d), a potent cytotoxic molecule to cells (reaction (7)).
Thus, it is possible that the eventual pathway leading to ischemia/reperfusion injury may arise from hydroxyl radicals or hydroxy radical-like species produced by peroxynitrite as a result of simultaneously increased superoxide anion and nitric oxide. Studies using cultured neurons suggest that both NMDA- and glutamate-induced neurotoxicity and neuronal damage due to hypoxia may be mediated by .NO (see, for example, Bredt and Snyder, Neuron, 8: 3-11, 1992 and Manzoni et al., Neuron, 8:653-662,1992).
Several drugs, aimed at blocking free radical-induced reperfusion injury, have been developed and tested in animals and humans. They can be categorized into two major types, namely, inhibitors and scavengers. For example, ganglioside GM-1 (which binds calmodulin and inhibits NOS activities) has been evaluated in acute ischemic stroke (see, for example, Lenzi et al., in Stroke, 5:1552-1558, 1994). However, treatment with GM-1 did not appear to alter patient survival. As another example, lubeluzole, a newly synthesized benzothiazole compound, is in phase II clinical trials for the treatment of acute ischemic stroke (see, for example, Diener et al., in Stroke, 27:7681, 1996). This drug inhibits glutamate-induced nitric oxide-related neurotoxicity by interfering with key mechanisms in the biochemical cascade that lead to ischemic tissue damage. Clinical trials are also in progress for several other glutamate antagonist drugs, but data have not yet been published (see, for example, Meldrum, in Current Opinion in Neurol., 8:15-23, 1995).
Currently, many pharmaceutical companies have turned their attention to the design and development of substrate or product analogue inhibitors of the nitric oxide synthase enzyme, NOS, in efforts to treat the overproduction of .NO in stroke and other ischemic/reperfusion conditions. For example, aminoguanidine, an NOS inhibitor, was shown to ameliorate the brain damage in cerebral ischemia (see, for example, Zhang et al., in Stroke, 27:317-323, 1996). Inhibition of NOS by NG-nitro-L-arginine decreased lipid peroxidation in the gerbil cerebral ischemia (see, for example, Caldwell et al., in Eur. J. Pharmacol., 285:203-206, 1995).
However, recent data show that the inhibition of NOS is detrimental to subjects. For example, rodent studies have shown that inhibition of the production of .NO causes intrauterine growth retardation and hind-limb disruptions in rats (see, for example, Diket et al., in Am. J. Obstet. Gynecol., 171: 1243-1250, 1994). Furthermore, the inhibition of NOS was found to cause myocardial ischemia in endotoxic rats (see, for example, Avontuur et al., Cir. Res., 76:418-425, 1995).
In contrast to the inhibitory approach described in the prior art to address the problem of free radical overproduction, the free radical scavenging approach also has been taken to reduce excessive reactive oxygen and nitrogen species in vivo. For example, tirilazad mesylate, a free radical scavenger, has been employed in clinical trials for the treatment of stroke patients (see, for example, Haley, in Stroke, 25:418-423 (1994)).
There is, however, still a need in the art for agents which effectively block free radical-induced reperfusion injury, without causing undesirable side effects.
In accordance with the present invention, there is provided a new class of drugs for therapeutic treatment of cerebral stroke and other ischemia/reperfusion injury. Thus, in accordance with the present invention, dithiocarbamates are linked to the surface of a macromolecule other than an antibody (e.g., albumin protein) under crosslinking conditions selected to preserve the dithiocarbamate either by using cross-linking reagents or by nonspecific binding to produce non-targeting polydithiocarbamate-macromolecule-containing derivatives and compositions containing such derivatives. The invention derivatives represent a new class of drugs for therapeutic treatment of cerebral stroke and other ischemia/reperfusion injury. There are numerous advantages of the invention polydithiocarbamate-macromolecule-containing compositions for ischemia/reperfusion therapy, including:
(i) providing multiple thiol groups, which are reducing equivalents that are known to react effectively with reactive oxygen species such as superoxide anion and hydroxyl radicals and with reactive nitrogen species such as nitric oxide to form S-nitrosothiol derivatives,
(ii) chelating and removing adventitious iron ions released from injured tissues to prevent oxidative damage (caused, for example, by iron-catalyzed oxygen radical reactions), and
(iii) forming, upon chelation with iron, a two-to-one [(dithiocarbamate)2-Fe] complex on the surface of the macromolecule. This complex further scavenges excess nitric oxide produced in inflamed tissues such as cerebral infarcts in ischemic stroke.
The simultaneous removal of reactive nitrogen species (such as nitric oxide) and reactive oxygen species (such as superoxide anion radical and hydroxyl radicals) should impede the pathway leading to the formation of peroxynitrite, reducing the generation of reactive hydroxyl radical-like species, as shown in reactions (5)-(7) above, and thus ameliorating ischemia/reperfusion injury.
In accordance with another aspect of the present invention, combinational therapeutic methods have been developed for the in vivo inactivation or inhibition of formation (either directly or indirectly) of species which induce the expression of inducible nitric oxide synthase, as well as reducing nitric oxide levels produced as a result of .NO synthase expression. Invention combinational therapeutic methods can be employed, for example, for the treatment of infectious and/or inflammatory conditions. Thus, the effectiveness of many therapeutic agents used for the treatment of infectious and/or inflammatory conditions can be enhanced by co-administration thereof in combination with the dithiocarbamate-containing nitric oxide scavenger(s) described herein.
Additionally, proton magnetic resonance imaging (MRI) techniques provide important information on images of regions of acute infarctions in cerebral ischemia in humans (see, for example, Warach et al., in Neurol., 42:1717-23, 1992). MRI techniques coupled with the use of contrast agents are being developed to assess cerebral perfusion after ischemic insults (see, for example, Fisher et al., in Ann. Neurol., 32:115-122, 1992). Because of its inherent paramagnetic properties, iron containing complexes of polydithiocarbamate-macromolecule-containing compositions according to the present invention should also be useful as a contrast enhancement agent for the measurement of blood perfusion in various organs including brain, heart, kidney and other vital organs and to assess the infarct area and volume in ischemic stroke and heart attack.
Thus, in accordance with another aspect of the present invention, magnetic resonance imaging methods have been developed for the measurement of cerebral and cardiac blood flow and infarct volume in ischemic stroke or heart attack situations. Such methods employ iron-containing complexes of a composition comprising a dithiocarbamate and a macromolecule as contrast agents. It has been found that conjugation of a dithiocarbamate and a macromolecule, as described herein, produces dithiocarbamate-macromolecule-containing compositions having both free radical scavenging and hemodilution beneficial effects in the treatment of ischemia/reperfusion injury.